S-bis(3-meth acrylate-2-hydroxypropyl)ether (BisS-GMA) was prepared. CQ (camphorquinone) of 1 wt % and DMAEMA (2-(dimethylamino)ethyl methacrylate) of 2 wt % were used in a photoinitiation system to initiate the copolymerization of the matrix resins. Distilled water contact angle measurements were performed for the wettability measurement. Degree of conversion, volumetric shrinkage, contraction stress and compressive strength were measured using Fourier Transformation Infrared-FTIR spectroscopy, the AccuVol and a universal testing machine, respectively. Within the limitations of this study, it can be concluded that the resin composites modified by bismethylene spiroorthocarbonate and BisS-GMA showed a low volumetric shrinkage at 1.25% and a higher contact angle. The lower contraction stress, higher degree of conversion and compressive strength of the novel dental composites were also observed.In this study, a novel dental composite based on the unsaturated bismethylene spiroorthocarbonate expanding monomer 3,9-dimethylene-1,3,5,7-tetraoxa-spiro[ These materials not only have superior physiochemical performance and manipulative qualities, but also acceptable appearance and favorable biocompatibility. They have the advantage of directly bonding to the tooth structure without the sacrifice of removing healthy tissues. As a result, light-curing resin composites are an area of research interest in many aspects –4.The age of modern dental composites starts with Bowen’s resin, 2,2-bis[4(2-hydroxy-3-methacryloylpropyloxy)phenyl]propane (Bis-GMA), which is the primary organic ingredient in nearly every commercial restorative resin because of the outstanding ability to form cross-links (which are stronger than linear polymers) during polymerization. Although the composite based on Bis-GMA has become vital for dental restoration due to its superior aesthetic quality, simple administration, and enhanced mechanical strength, there are still problems. An important issue of the monomer phase is that it undergoes polymerization shrinkage. Polymerization shrinkage  and subsThe volumetric shrinkage of resin composites during polymerization arises from two different factors: the van der Waals distance between the monomer molecules is replaced by a covalent bond during polymerization, and the intermolecular distance between the polymer chains becomes smaller than that between the monomers. Numerous studies aimed at developing resin composites with a low level of curing shrinkage have been performed –11. ManyS-bis (3-methacrylato-2-hydroxy propyl) ether) was synthetized [A goal of our long-time work has been to develop monomers that can mitigate polymerization stress and shrinkage in dental resin systems. In our previous study, BisS-GMA /T resin composite before and after cured. In comparison with the dental matrix resins before and after cured, aromatic C=C (1590 cm−1) peak almost kept invariable, while the aliphatic C=C (1635 cm−1) was obviously reduced, which meant that the number of the residual C=C (1635 cm−1) decreased, indicating the occurrence of the copolymerization reactions. Meanwhile, tetraoxaspiro C–O absorbance at 1212 cm−1 was significantly reduced, which means the occurrence of the ring-opening reactions.Copolymerization reactions and the ring-opening reactions of the matrix resins were measured by FTIR. p < 0.05). The DC of (BMSOC + BS)/T tested after cured 24 h was significantly increased compared with the conventional dental matrix resins Bis-GMA/TEGDMA (B/T) (p < 0.05). The results suggest that BisS-GMA is more effective than Bis-GMA when referring to the degree of conversion, which is consistent with prior work. The explanation of superiority of BisS-GMA originates from the different structure between the two monomers. The oxygenic atom of O=S=O bond in BisS-GMA has a stronger electronegative power than methyl when connected with tertiary carbon in Bis-GMA, so the rate of reaction is speeded up and the degree of conversion is increased. Additionally, the presence of vinyl functionality in the BMSOC increased the reactivity with the bifunctional BisS-GMA. Degree of conversion is directly related to polymerization shrinkage stress [The degree of conversion which was calculated from the equivalent aliphatic/aromatic molar ratio from polymerized and unpolymerized samples is presented in e stress . In addie stress . Higher e stress . The nov2.2.p < 0.05). The addition of SOC increased significantly the contact angle (p < 0.05). Gan et al. reported that the contact angle data of the commercially available resin-based composites ranged between 31.5° and 64.5° under distilled water [Measurement of the contact angle at the solid-air-liquid meeting point is a widely known technique used to investigate wettability of solid substrates. The means and standard deviations of the contact angles of the studied groups are summerized in ed water . The coned water . Seconda2.3.p < 0.05) lower shrinkage values in comparison to the corresponding Bis-GMA-containing resins. Values for BMSOC-containing test materials exhibited statistically significant (p < 0.05) lower shrinkage values than the corresponding BMSOC-free resins. The contraction stress of the resin composites corresponded to the result of shrinkage. (SOC + BS)/T resins exhibited the lowest shrinkage and contraction stress (p < 0.05).p > 0.05).CS testing is considered to be one of the most reliable methods to determine the mechanical strength of dental composites. The mechanical properties of CS of the resin composites containing 70 wt % of inorganic filler were examined. The values reported in MPa were the average number from the measurement of 12 replicate specimens, and computer generated automatically. It was observed that the groups of (SOC + B)/T and (SOC + BS)/T composites had significantly increased with respect to B/T and BS/T composites respectively. In addition, the CS of groups containing BisS-GMA had a higher value than Bis-GMA-containing composites. The CS was significantly different between any two groups of composites except BS/T and (SOC + B)/T : 3097 cm−1 (C=C), 1212 cm−1 (tetraoxaspiro C–O). 1H NMR  δ: 4.44 , 4.93 . Melting point: 79.8–81.2 °C. The result is consistent with that previously reported [The target product was white solid and characterized by FTIR, analyses . Yield: reported .3.2.1H NMR according our previously study [2 were obtained from Sigma-Aldrich Co., Milwaukee, WI, USA. The particle size and shape of the filler SiO2 were larger than 230 mesh. Before being mixed in matrix resin, the SiO2 micro powder was modified by the coupling agent KH-570 [2 (30 mL:1 g) was ultrasonically dispersed, and then the modifying agent (KH-570) was added dropwise into the dispersion under continuous agitation. The mixture was stirred for 8 h in the boiling state to obtain the modified SiO2 which could be mixed up with the organic matrix resin. The chemical structure of materials used in the composites is depicted in BisS-GMA was synthesized at Chengdu institute of organic chemistry, the University of the Chinese Academy of Sciences. The molecular structure of BisS-GMA was finally confirmed by FTIR and ly study . Conventt KH-570  /T and (SOC + BS)/T. Composition of each group has been summarized in Four groups of dental matrices with TEGDMA (30 wt %) which contained Bis-GMA/BisS-GMA and/or BMSOC were prepared as follows: Bis-GMA, BisS-GMA, BMSOC/Bis-GMA (3:7), BMSOC/BisS-GMA (3:7), with 1 wt % CQ as an initiator and 2 wt % DMAEMA as an accelerator. Mixing with inorganic fillers SiO3.4.The wettability of the samples was assessed by measuring the contact angles of distilled water on composite resins with a contact angle measuring system . The contact angle was defined as the angle at which the liquid interface met the solid surface of the composite disc at four points on each sample, and the mean of the points was reported as the contact angle of each sample. The surface of the drop was monitored constantly, and the contact angle was measured just after 20 s, when the droplet was stabilized .3.5.2 was used. The uncured and cured samples of the composite were placed above a horizontal zinc crystal. Measurements were made 24 h after photo activation (n = 5). Absorption spectra were recorded in a transmission mode using 16 scans at a resolution of 4 cm−1. The ratio of maximum absorption was determined using the base line method considering the peaks of 1635 cm−1 for aliphatic, and 1590 cm−1 for aromatic, chains. The degree of conversion was calculated from the equivalent aliphatic/aromatic molar ratio from polymerized (P) and unpolymerized (U) samples using the formula:Degree of conversion (DC) was measured by Fourier Transformation Infrared-FTIR  (Prestig3.6.n = 7) was shaped into a semisphere (with volumes averaging approximately 7 μL) and placed on a polytetrafluoroethylene pedestal in front of a CCD camera within the device. Special software was employed for the acquisition and processing of images. The uncured sample was allowed to rest for 1 min to eliminate the influence of slumping on the measurement and was later exposed to 40 s of light curing, with the curing unit tip positioned 1–2 mm from the specimen. The volume shrinkage image was recorded five min after light-curing in order to lower the temperature to room temperature.The VS values of the composite specimen were measured using a video-imaging device  (AccuVol3.7.n = 7) was light-cured for 40 s. The axial contraction stress development was recorded continuously from the start of curing until 30 min had elapsed.The polymerization-induced contraction stress was determined with a universal testing machine  with a fixture holding the composite material on a glass plate parallel to the flat surface of the steel cylindrically shaped rod with a six mm diameter ,39. A stp < 0.05 was considered statistically significant.All measurements above are presented as mean ± standard deviation and compared with the one-way ANOVA tests and LSD tests using SPSS  17.0 software. A value of 3.8.n = 12) were prepared from each experimental material using copper cylindrical molds with dimensions of 6 mm (in diameter) × 4 mm (in height). The composite was cured for 40 s in three consecutive and overlapping sections to ensure the most complete polymerization possible. The specimens were stored in distilled water in an incubator  at 37 °C for seven days and then were subjected to compressive testing using the Universal Testing Machine  with a load cell of 50 N at a crosshead speed of 1 mm/min, in accordance with ADA specification No. 27. The results were recorded in megapascals (MPa).Bar-shaped specimens (4.The aim of this study was to investigate the modification of Bis-GMA/TEGDMA dental resin composites with unsaturated bismethylene spiroorthocarbonate expanding monomer and BisS-GMA matrix monomer. The degree of conversion, volumetric shrinkage, contraction stress and microleakage properties were studied. The results indicated that the addition of unsaturated bismethylene spiroorthocarbonate expanding monomer and BisS-GMA matrix monomer mixtures reduced the polymerization-shrinkage and contraction stress in comparison with conventional methacrylate-based composites. Meanwhile, the increased degree of conversion, higher contact angle and improved mechanical properties of the novel resin composites were acquired, which make these new types of resin composites worthy of further study. Ongoing investigations into their physicochemical properties, biocompatibility and the ring-opening mechanism involved, are currently in progress and will be reported in due course.


So far, only one study investigated the circulating Tfh cells increased in a subset of SLE patients. Since relatively little is known about the Tfh cells in rheumatoid arthritis (RA) patients, in this study, Tfh-cell frequency, related cytokine IL-21, and transcription factor Bcl-6 were investigated in 53 patients with RA and 31 health controls. Firstly, we found that the frequency of CD4+CXCR5+ICOShighTfh cells was increased significantly in the peripheral blood of RA patients, compared with that in healthy controls. It is known that Tfh cells are critical for directing the development of an antibody response by germinal centers B cells; secondly, we observed that the Tfh-cell frequency is accompanied by the level of anti-CCP antibody in RA patients. Furthermore, expression of Bcl-6 mRNA and plasma IL-21 concentrations in RA patients was increased. Taken together, these findings have shown that the increased frequency of circulating Tfh cells is correlated with elevated levels of anti-CCP antibody, indicating the possible involvement of Tfh cells in the disease progression of RA.Follicular helper T (Tfh) cells are recognized as a distinct CD4 With the help of T cells, activated B cells migrate into lymphoid follicles of lymphoid organs and form germinal centers (GCs) [+ T cells in regulating B cells proliferation and antibody production especially in the GC structures [Rheumatoid arthritis (RA) is a chronic and symmetric polyarticular arthritis that primarily affects the small diarthrodial joints of the hands and feet . The salrs (GCs) . Within rs (GCs) , 4. Currructures .+ T subset, have been found to be present in GCs [in vivo [Recently, follicular helper T (Tfh) cells, a novel CD4t in GCs , which r[in vivo . Tfh cel[in vivo , 9. High[in vivo . It beco[in vivo . However+CXCR5+ICOShigh Tfh cells was increased in SLE patients, which prompted us to examine the frequency of circulating Tfh cells in the peripheral blood of RA patients and its correlation with autoantibody production. In this study, the increased frequency of CD4+CXCR5+ICOShigh circulating Tfh cells was detected in RA patients, which was positively correlated with high levels of serum anti-CCP antibody. Thus, these results have indicated the possible involvement of Tfh cells in the pathogenesis of RA.An elegant study by Simpson et al.  has receA total of 53 RA patients and 31 health controls were enrolled in the present study. Fifty-three newly diagnosed RA patients without treatment from 2009 to 2010 at the Affiliated People's Hospital of Jiangsu University were included in this study. RA patients fulfilled the 1987 revised criteria of the American College of Rheumatology (ACR) . Thirty-+ T cells were purified from PBMCs by FITC-conjugated anti-human CD4 mAb and anti-FITC microbeads  according to the manufacturer's instructions.Plasma was collected through centrifugation and stored at −80°C for measurement of cytokine levels. PBMCs were isolated by standard Ficoll-Paque Plus density-gradient centrifugation for analysis by flow cytometry. CD4For phenotypic analysis, fluorescein isothiocyanate (FITC)-ICOS, phycoerythrin (PE)-CD4, and PE-Cy5-CXCR5 monoclonal antibodies against human-cell surface were purchased from eBioscience . All the staining was performed according to the manufacture's protocol. The stained cells were then analyzed using the BD FACScalibur system and CELLQUEST software.+ T cells, CD4+ T cells were incubated in complete RPMI1640 culture medium in the presence of 50 ng/mL phorbol myristate acetate (PMA). After 4 h of culture at 37°C under 5% CO2, the cells were collected and centrifuged at 500 g for 5 min. After being washed in phosphate-buffered saline (PBS), TRIzol reagent was added. Total RNA was extracted from CD4+ T cells with TRIzol reagent and cDNA synthesized according to manufacurer's instructions . Each gene was amplified in triplicate, and cDNA concentration differences were normalized to β-actin [For the detection of IL-21 mRNA of CD4 β-actin . Primer Level of plasma IL-21 was done using ELISA for human IL-21 . All determinations were performed by duplicate, and the lower detection limits for IL-21 were 31 pg/mL. The plasma from RA patients was tested for the presence of anticyclic citrullinated peptide (anti-CCP) antibody by ELISA. All samples were analyzed in duplicate using the average of the optical density (OD) values to calculate concentrations.P < 0.05 as determined by student's t-test using SPSS13.0. Correlation of nonparametric paired data was tested using Spearman's rho, and the significance was evaluated using the t statistic.Data were presented as mean ± standard deviation in the text and figures. Statistical differences were considered to be significant at a value +ICOS+T cells was increased in RA patients. We first analyzed the frequency of CD4+ICOS+ and CD4+ICOShigh T cells in PBMCs from RA patients and healthy controls by flow cytometry. Figures +ICOS+ and CD4+ICOShigh T cells in RA patients were higher than those in healthy controls (P < 0.001). CXCR5 has enabled the Tfh cells to enter the follicles during T-cell-dependent immune responses. In both mice and humans, there have been shown that CXCR5+and ICOShigh are important phenotype for CD4+Tfh cells. We use flow cytometry to analyze the frequency of the circulating Tfh cells in PBMCs from RA patients and healthy controls. A significantly increased frequency of CD4+CXCR5+ and CD4+CXCR5+ICOShigh circulating Tfh cells was detected in RA patients, compared with that in healthy controls . Our data indicated that increased fraction of the circulating Tfh cells is apparent in a subset of RA patients.Because the inducible costimulatory molecule (ICOS) is a T-cell activation marker, and it has been reported that the frequency of CD4 Figures   and frequency of the circulating Tfh in RA patients. According to the titer of anti-CCP antibody, RA patients were divided into two groups: anti-CCP antibody-positive group and anti-CCP antibody-negative group. The frequency of the circulating Tfh cells showed a positive correlation with anti-CCP antibody-positive group in plasma   (P < 0.05)   . IL-21 i < 0.01) . At same < 0.05) . Additio 0.9100) .+CXCR5+ICOShigh T cells resembles circulating Tfh cells and tonsillar Tfh cells [+CXCR5+ICOShigh Tfh cells was increased significantly in RA patients, compared with healthy controls. The percentage of CD4+ICOS+ cells and CD4+ICOShigh cells in RA patients were also higher than those in healthy controls. Our data provide first evidence that the circulating Tfh cells were increased in RA patients, suggesting that Tfh cells may be involved in the pathogenesis of RA.Tfh cells are a class of regulatory T help cells that specialize in the cognate control of antigen-specific B-cell immunity , differefh cells . In thisIt is known that Tfh cells are critical for directing the development of an antibody response by GC B cell. The presence of autoantibody in the serum is a typical phenomenon for RA patients. Rheumatoid factor (RF) was observed originally by Franklin et al. in 1939 –22. We a+ T cells of RA patients were increased significantly. Bcl-6 transcription factor is selectively expressed by Tfh cells [+ T cells of RA patients by RT-PCR.Bryant et al.  have foufh cells , 25. Thefh cells . Our datIn conclusion, our data provide evidence of increased circulating Tfh cells in RA patients, suggesting that the increase of the circulating Tfh cells may be associated with the development of RA. However, it is critical to further determine the mechanisms of controlling the production and activation of Tfh cells.


This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45±0.07 mm2 in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III . These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.In models of  Although reports on ghrelin neuroprotective effects are consistent, it is reported that ghrelin can exert some anticonvulsant effects in pentylenetetrazole-treated rats, in the penicillin model of cortical ictogenesis and also in models of chemoconvulsive SE, such as in the case of pilocarpine or kainate treatment , weighing 230–250 g were used. All the experiments were performed in accordance with the European Directive 2010/63/EU and approved by the Italian Ministry of Health (DM 126/2011 - B). All efforts were done to minimize the number of animals and their sufferance. Pilocarpine  was used to induce SE. The pilocarpine injection was preceded by methylscopolamine  to prevent the peripheral effects of cholinergic stimulation In vivo video ECoG was performed in a subset of rats  during pilocarpine-induced SE. Electrodes were implanted as previously described Pilocarpine-treated rats  and non-epileptic control rats (n = 8) were anesthetized with chloral hydrate  4 days after the injections and perfused through the ascending aorta, using 100 ml saline followed by 100 ml 4% paraformaldehyde and picric acid (0.3%) dissolved in 0.1 M phosphate buffer (pH 6.9). Brains were kept overnight in the same fixative at 4 °C and cryoprotected by immersion in 15% and 30% sucrose-phosphate buffer solutions. They were then frozen and cut horizontally with a freezing microtome in serial 50 µm-thick sections from bregma levels −9.1 mm to −5.1 mm 2).We stained degenerating neurons in adjacent sections to those used for immunohistochemistry, using Fluoro-Jade B  as fluorochrome. Brain sections  were washed in PBS, mounted on gelatin-coated slides and dried overnight (37 °C). Sections were progressively hydrated by immersion in decreasing ethanol solutions (3 min in absolute ethanol and 1 min in 70% ethanol) and treated with 0.06% potassium permanganate for 15 min. After a 1 min wash in distilled water, sections were stained in a 0.001% Fluoro-Jade B solution containing 0.1% acetic acid. Sections were then washed in water, dried on a pre-warmed hotplate at 37 °C, dipped in xylene and covered with mounting medium  2) of GFAP and laminin immunoreactivity were acquired in CA3 by an Axioskop microscope  using a Sony CCD-IRIS BW video camera. GFAP lesions, expressed in mm2 for each animal, were delimited as previously illustrated 2. A similar procedure was used for the ET-1 immunostaining in CA3. Neuronal cell profiles were obtained as previously illustrated 2 fields within the hilus and layer III of the medial entorhinal cortex of each serial section and expressed as cell densities (n/mm2) by considering a minimum cut-off value of 7 µm. In the CA1 region of the hippocampus close to the subiculum  as previously described P<0.05 was selected as threshold for significant differences between the means.Statistical comparisons were performed by one-way analysis of variance (ANOVA) and followed by the Fisher's least significant difference (LSD) test . Values are presented as mean±SEM and a In all rats, pilocarpine successfully induced seizures and SE assessed by the experimenter  and vide1a agonists, it was significantly reduced in both ghrelin and JMV-1843 treatment groups  and 57% in the JMV-1843 group   .1a agonists would also affect the laminin immunoreactivity in the hippocampus  in the saline group of pilocarpine-treated rats  rats.To evaluate possible protective effects of GHS-Rhe hilus , a regioted rats . Hilar nP<0.01) in the saline group of pilocarpine-treated rats . However, neuronal cell density was significantly (P<0.05) higher (+60−70%) in the JMV-1843 group compared with saline- or ghrelin-treated rats . A similar decrease was observed in GHS-R1a agonist-treated rats: NeuN-positive cells were decreased, respectively, to 77% in ghrelin (P<0.05 vs controls) and to 67% of control levels in JMV-1843 (P<0.01) groups. Contrary to what we observed in the hilus and entorhinal cortex, JMV-1843 did not have any protective effect in the CA1.A different scenario emerged from the analysis of neuronal cell densities in the hippocampus proper. The highly packed distribution of neurons in the CA1 and CA3 regions did not allow a similar analysis to the one performed in the hilus of dentate gyrus and medial entorhinal cortex layer III. Thus, we sampled at higher magnification the CA1 sector proximal to the subiculum  and the P<0.05) in saline-treated rats. In rats treated with GHS-R1a agonists, neuronal cell densities were also decreased but did not reach significant statistical difference . A similar scenario was found also in the medial entorhinal cortex, layer III (P<0.01) and ghrelin (P<0.05) groups.NeuN has been used often as a specific marker of neuronal cell loss and it provided coherent results both in pilocarpine he hilus , JMV-184ayer III , where t1a found to exert protective actions against neuronal cell death in various experimental models, including hypoxia and glucose deprivation 1a agonist JMV-1843 in the pilocarpine model of SE, in which both apoptosis Ghrelin is an endogenous ligand of GHS-Rlimitans from the basal lamina of blood vessels, leading to disruption of the blood-brain barrier Initially, we evaluated the effects of ghrelin and JMV-1843 on the vascular lesion that occurs in the CA3 hippocampal region after pilocarpine treatment 1a agonists on blood vessels in CA3. We have previously shown that an intracerebral injection of ET-1 is able to reproduce the changes of laminin and GFAP immunoreactivities observed in CA3 after SE We also tried to define a possible mechanism for the observed protective effects of GHS-R1a, namely JMV-1843. JMV-1843 is known to possess a better pharmacokinetics than ghrelin We evaluated neuronal integrity by counting neurons stained by anti-NeuN antibodies. Although NeuN is universally used as a marker for the evaluation of neuronal cell damage in different brain lesion models 1a agonists results in beneficial effects in pilocarpine-treated rats exposed to SE. The neuroprotection afforded by ghrelin and JMV-1843 was completely independent of anticonvulsant effects, as shown by the ECoG recordings. JMV-1843, which is a peptidomimetic orally active ligand of the GHS-R1a, was more effective than ghrelin. Interestingly, this difference in protection was associated with repression of ET-1 synthesis in perilesional cells, suggesting that this phenomenon is involved in neuroprotection during prolonged seizure exposure.In conclusion, we show that pretreatment with GHS-R


GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s−1 vs 47.86±2.78 s−1) is balanced by an increased glucose affinity (S0.5 = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing.Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding  In addition, and also in contrast to other hexokinase isoforms, glucokinase activity might be modified by protein interaction partners that can modulate the catalytic activity and the intracellular distribution of the enzyme in beta-cells and/or hepatocytes (for review see Glucokinase (GK) plays a key role as the pancreatic beta-cell glucose sensor by integrating blood glucose levels and glucose metabolism with insulin secretion GCK gene cause different monogenic glycaemic disorders  was prepared as described previously +-coupled assay with glucose-6-phosphate dehydrogenase, as described previously 2, 1.4 mM ß-mercaptoethanol, 0.1% BSA, 5 mM ATP-MgCl2, 1 mM NADP+, and 1 U/ml glucose-6-phosphate dehydrogenase. Determination of kinetic parameters and thermal stability tests were performed as in 0.5 value for GST-GK wild-type and mutant proteins , GST-GK(p.Gly223His), GST-GK(p.His416Arg) and GST-GK(p.Ala449Thr), respectively. The relative activity index was calculated as previously 50) were derived from Dixon plots 2; 2.5 mM MgCl2; 5 mM ß-mercaptoethanol, 1 U/ml glucose-6-phosphate dehydrogenase and 61 nM glucokinase Glucokinase activity was measured spectrophotometrically on a spectrophotometer Uviconxl (Secoman), using an NADPPurified human GKRP was kindly provided by KJ Brocklehurst . The effect of GKRP on glucokinase activity was determined as described One unit of GK is defined as the amount of enzyme that phosphorylates 1 µmol of glucose per min at 30°C.P values of <0.05 were considered statistically significant. The structural analysis of mutations was carried out using the crystal structure of human GK GK activator LY2121260 was kindly provided by Elli Lilly and Co, Indianapolis, IN, USA. A stock solution of 1 mM LY2121260 was prepared in 12.5 mM Hepes pH 7.4 with 80% DMSO. Results are shown as means ± SEM, and statistical significance was analysed by the two-tailed Student's t-test. S. cerevisiae strain Y187 as described previously Two-hybrid studies were performed in GCK heterozygous missense mutation was identified in each family. Three mutations  had been previously reported as a cause of MODY E. coli. Kinetic parameters of purified fusion proteins are shown in a) values. Among these mutations p.Gly223Ser and p.His416Arg significantly affected all the kinetic parameters. Both mutations decreased the affinity of GK for both substrates glucose and ATP, and also decreased the catalytic constant (Kcat) and the Hill coefficient (nH) of the enzyme. In contrast, mutation p.Ile130Thr, which also decreased the Kcat and the nH, increased the affinity of GK for ATP without modifying the affinity for glucose.Recombinant wild-type GST-GK and mutants GST-GK(p.Ile130Thr), GST-GK(p.Asp205His), GST-GK(p.Gly223Ser), GST-GK(p.His416Arg) and GST-GK(p.Ala449Thr) were prepared in in vitro activity index slightly higher than the wild-type protein, although this difference was not statistically significant (p = 0.11). However, and despite this apparent normal activity level, this mutation affects all the kinetic parameters of GK individually. The mutant protein GST-GK(p.Ala449Thr) lacked the cooperativity for glucose, as shown by a significantly decreased Hill coefficient. The Kcat of this mutant was reduced to 7% of the wild-type value and the affinity for ATP was decreased more than 3-fold. In contrast, and unusually for MODY2 mutations, affinity for glucose was strongly increased in about 5-fold. The protein stability of mutant GST-GK(p.Ala449Thr) was tested at different temperatures and compared to that of the wild-type fusion protein. Mutant GST-GK(p.Ala449Thr) had an 50 value, is significantly affected for the GK(p.Gly223Ser) mutant with respect to the wild-type enzyme. In contrast, mutation p.Ala449Thr did not affect the inhibition by NAG but decreased the I50 for MH and palmitoyl-CoA by about 35 and 26%, respectively.To further investigate the mechanism by which mutation p.Ala449Thr increased the affinity of GK for glucose, we tested the effects on both mutant and wild-type enzymes, of some competitive GK inhibitors such as N-acetylglucosamine (NAG), mannoheptulose (MH), palmitoyl-CoA, as well as the glucokinase regulatory protein (GKRP). Palmitoyl-CoA and GKRP bind to distinct allosteric sites, whereas glucose analogues NAG and MH bind to the catalytic site of the open and closed conformations, respectively 0.5 but produced a slight recovery of Kcat up to about 20% of the normal wild-type value.Finally, we studied the effects of the GK synthetic allosteric activator LY2121260 on the kinetic parameters of GST-GK(p.Ala449Thr) and compared them to those on the GST-GK wild-type protein . For theGCK mutations identified in probands with familial, mild fasting hyperglycaemia. The biochemical characterization of these mutations has shown that all of them impair normal glucokinase activity. The molecular analysis of these mutations revealed the particular defects caused in GK enzymatic activity.In this report we describe the functional analysis of five super-open conformation. Following glucose binding, the enzyme undergoes a conformational change leading to, first, an intermediate open conformation and then, a fully active closed conformation, which binds ATP. The transition between the super-open and closed conformations involves the rotation and approximation of the small domain to the large one. The existence of GK in different conformations allows two catalytic cycles, fast and slow, that explain the sigmoidal response to glucose The structural data obtained from the free and substrate/activator bound GK crystals showed that this enzyme has a small and a large domain separated by a deep cleft where substrates bind ie low affinity for glucose and cooperativity, are lost in this mutant protein.The activity index obtained from the kinetic analyses of these mutants might be sufficient to explain the pathogenic effect of mutations p.Asp205His, p.His416Arg, p.Gly223Ser and p.Ile130Thr, since there is a direct relationship between this parameter and the glucose-stimulated insulin secretion threshold in a heterozygous mutant 0.5h) similar to that of the wild-type (2.44±0.39 vs 2.29±0.23). In the structural model of GK, the side chain of alanine or threonine at position 449 is projected to the solvent in the super-open inactive conformation (0.5 to a similar extent suggests non-productive substrate binding or blockage in substrate release, which could be due to an impairment of this mutant protein to return to its open form in order to release G6P and ADP Ala449 is located in the α13 helix which is the C-terminal region of glucokinase . The α13ormation . In contormation . The larIn summary, the results of the biochemical analysis of these different MODY2-causing mutations illustrate the importance of the fine tuning of GK and its role in glucose sensing, and provide further insights into the role of the catalytic and allosteric activator sites in the regulation of the activity of this enzyme.


Pramipexole, the most commonly prescribed dopamine agonist worldwide, meanwhile serves as a reference substance for evaluation of new drugs. Based on numerous clinical data and vast experiences, efficacy and safety profiles of this non-ergoline dopamine agonist are well characterized. Since October 2009, an extended-release formulation of pramipexole has been available for symptomatic treatment of Parkinson's disease. Pramipexole administration can be cut down from three times to once a day due to the newly developed extended-release formulation. This is considerable progress in regard to minimizing pill burden and enhancing compliance. Moreover, the 24 h continuous drug release of the once-daily extended-release formulation results in fewer fluctuations in plasma concentrations over time compared to immediate-release pramipexole, given three times daily. The present study summarizes pharmacokinetics and all essential pharmacological and clinical characteristics of the extended-release formulation. In addition, it provides all study data, available so far, with regard to transition and de-novo administration of extended-release formulation for patients with Parkinson's disease. It further compares efficacy and safety data of immediate-release pramipexole with the extended-release formulation of pramipexole. Parkinson's disease is a chronic neurodegenerative illness that requires regular medication unlimited in time. Parkinsonian patients will comply much more readily to simple therapeutic regimens with fewer applications than to treatments with more frequent drug administration . PharmacA prolonged-action drug permits the steady release of the active ingredient over 24 hours preventing the active concentration from continuously surging on and surging off as commonly encountered with multiple applications of the substance. An unvarying, efficient level by continuous release over 24 hours will help avoid losses during the course of the day. This is nurtured by the hope for better control of motor symptoms during the day and for less nocturnal and matutinal complaints, akinesia for instance, aside from late motor complications in the long run. At the same time, undesired effects might be abated since peak concentrations no longer occur. Pramipexole, the dopamine agonist that had been a non-depot formula so far, which patients had to take tid (3x) daily, could be turned into a controlled-release drug to be administered once daily. This paper gives an overview regarding the development and pharmacology of the extended-release form of pramipexole, and it summarizes the available clinical data on depot pramipexole in the treatment of early and advanced Parkinson's disease.Pramipexole  is a selective dopamine D2 receptor agonist, approved since 1998. Pramipexole is indicated for the symptomatic treatment of Parkinson's disease (idiopathic Parkinson's syndrome), either alone (without levodopa) or in combination with levodopa, that is, during the entire progress of disease up to the advanced stage. 2 subfamily. This substance shows a 7- to 10-fold higher affinity to D3 (Ki = 0.5 nM) than to D2 receptors (Ki = 3.9 nM). Pramipexole acts on presynaptic as well as on postsynaptic receptors [3 and D2 type, thereby reducing the synthesis and synaptic release of dopamine. Effects on postsynaptic receptors are only observed at higher doses, and they are marked by prolonged latency periods. With reduced dopamine release due to loss or damage of the presynaptic termini, however, the postsynaptic D2 and D3 receptors are additionally and immediately stimulated. Pramipexole is a full nonergoline dopamine agonist with selective affinity for dopamine receptors of the D2 and D3 receptors of the direct and indirect striatofugal nerve tracts. Here, pramipexole corrects the increased inhibitory impact of the direct pathway on motor activity by enhancing the direct transmission (through D3 receptors) and reducing the indirect transmission (through D2 receptors) [Pramipexole mediates its therapeutic action on motor deficits in Parkinson's disease via the postsynaptic Dceptors) .Until 2009, immediate release pramipexole tablets had been offered at a dosage of 0.125 mg, 0.25 mg, 0.5 mg, and 1.0 mg salt . Owing to their half-life of 8–12 hours, these immediate-release pramipexole tablets are taken tid in 3 equal doses to ensure the daily requirement. It has been an ardent desire of patients, doctors, care persons/family members to reduce the number of applications to a once-daily regimen. Other goals in the development of a controlled-release pramipexole formula were the simple 1 : 1 conversion from immediate-release pramipexole to extended-release pramipexole tablets overnight, a similar or improved peak/trough fluctuation, comparable plasma concentrations and comparable efficacy between the two formulas. Another significant objective was preventing the sudden and uncontrolled release of the active agent by all means (= “dose dumping”) under fasting conditions, after the ingestion of food and also of alcohol, which could lead to increased adverse effects. The drug substance pramipexole possesses numerous properties that encumbered development of a prolonged-release formula . Due to Several hundreds of pharmaceutical formulas were developed in view of these difficult challenges. There are so-called in vitro dissolution tests to test the behavior of prototypes. As depicted in All of these seven prototypes were also tested in a clinical phase I study. In a crossover study with in healthy male subjects , the phamax,ss), in the steady state (day 4 of each extended-release pramipexole therapy), that does not exceed the one obtained after immediate-release pramipexole t.i.d., and a minimum plasma concentration  not less than the one observed after the immediate-release formula applied t.i.d, a maximum plasma concentration (Ca peak/trough fluctuation (PTF) over 24 hours that is smaller than or comparable to the one following immediate-release pramipexole,a bioavailability not less than ≥75% in the steady state that compares with the immediate-release formula,no incidence of irregular release (“dose-dumping” defined as a not more than 80% dose absorption within 4 hours after application). One matrix tablet  with the chosen formula was established in the course that was able to predict a complete plasma concentration profile in humans by in vitro solubility charts  with ade0–24,ss). There were no further relevant differences in the maximum  and minimum concentrations . No significant differences versus food ingestion on an empty stomach [In a relative study on bioavailability, pharmacokinetic parameters were compared after the highest daily dose of 3.15 mg , administered as extended-release pramipexole once daily (q.d.) or as immediate-release pramipexole, 3x daily (t.i.d.) 1.05 mg base. The impact of food on the pharmacokinetics of extended-release pramipexole was additionally studied. The results were indicative of equivalent pramipexole plasma concentrations in the time span defined as surface on the chart -2-amino-4,5,6,7-tetrahydro-6-propylamino-benzothiazole dihydrochloride monohydrate equivalent to 0.26, 0.52, 1.05, 1.57, 2.1, 2.62, or 3.15 mg pramipexole base.Pramipexole is the most commonly prescribed dopamine agonist worldwide and may well be considered the reference substance in the evaluation of new agents. Many controlled studies have been published; the efficacy and the efficiency activity are well documented . Extendechange from immediate-release pramipexole to depot pramipexole,effectiveness of depot pramipexole in the early stage of disease,effectiveness of depot pramipexole in advanced Parkinson's disease. II and III were rated noninferior. 84.5% were successfully changed overnight (87 out of 103 patients). The proof on noninferiority failed although extended-release pramipexole turned out even better per UPDRS and CGI-I , responder rate (87. 4 versus 78.8%)  . There wA second switch study was conducted by Mizuno et al. in Japan , also maIn sum, the switch was uneventful in approximately 85% of the patients. The remainder did not complain about major side effects; they even indicated a better response under immediate-release pramipexole.The most valuable study to address this question was conducted by Poewe et al. . This woAnother important interim analysis in this type of patients is owed to Salin et al. . In thisParallel data on response were also collected. PGI interestingly enough revealed a slight superiority of the extended-release formula compared to immediate-release pramipexole in week 18 and 33 (45.7/42.9 versus 35.5/41.9%), evaluation of CGI disclosed a superiority of the immediate-release formula in week 18 and 33 (64.5/51.6 versus 46.9/40.6%).n = 99): the group taking extended-release pramipexole (n = 99) scored 7.4 points and the placebo group (n = 42) 2.7 points. Response rates were determined by GCI-I and PGI-I and were indicative of improvements in week 18 and 33 for extended-release pramipexole in 37/35.6%. The result was 48/23.8% under immediate-release pramipexole and 18/12% under placebo.Another analysis on extended-release pramipexole in the early stage of PD was published by Hauser et al. . EnrolleThe authors concluded that both pramipexole formulas were safe and superior to placebo, aside from being well tolerated. The efficacy of extended-release pramipexole was said to be similar to immediate-release pramipexole with comparable adverse effects.n = 385). Extended-release pramipexole proved superior after 18 weeks, reflected by 4.9 points (−11.0 versus 6.1) in UPDRS and 0.7 (−2.1 versus 1.4) hours off time as opposed to placebo. There were no relevant differences between immediate-release pramipexole and extended-release pramipexole. The great effect of placebo was astonishing. Giddiness and vomiting occurred less under extended-relapse pramipexole than under immediate-release pramipexole.The most significant study on extended-release pramipexole in the advanced stage of Parkinson's disease was carried out by Schapira et al. . This exThe aforementioned studies confirm preclinical data. Immediate-release pramipexole and extended-release pramipexole only vary in releasing the active agent from the tablet. The substance itself is unchanged, meaning there is an identical receptor profile, identical efficacy, and identical receptor binding. The half-life of the agent is also the same, but the continuous release from the depot tablet results in an overall prolonged plasma half life. Any of the accepted statements linked to immediate-release pramipexole also apply to extended-release pramipexole-except for the daily doses required. Extended-release pramipexole can be used in both early and late Parkinson's disease. A switch can take place overnight, the ratio being 1:1, for example, immediate-release pramipexole 3 × 0.7 mg (base) are consistent with 2.1 mg extended-release pramipexole. In most cases, the patient is not going to be affected by this switch. More efficaciousness, increased dyskinesias or undesired effects are not to be expected. In a few cases, the effect may be less, which would require a dose adjustment. Some patients appreciate the stimulating effect of immediate-release pramipexole with a rapid afflux and higher peak and for this reason, they prefer fast-release pramipexole. So far, there have been no insights regarding any increased individual side effect owing to the use of extended-release pramipexole. Reduction of plasma peaks might even result in decreased side effects. The once daily application constitutes the essential and cardinal advantage of the extended-release formula. This is definitely going to improve compliance, and to ameliorate disease control. The argument that the patient gets levodopa 3 times a day anyways does not hold, since levodopa is taken food dependent ; a 3x dSince the introduction of extended-release pramipexole, and in consideration of pharmacologic and clinical aspects, it has been recommended putting the patient on extended-release pramipexole in a new approach. All patients who have been under immediate-release pramipexole may furthermore be switched to extended-release pramipexole overnight. The inauguration of extended release-pramipexole is to be regarded as another important option in the drug treatment of Parkinson's disease.


Muscle growth depends on the fusion of proliferate satellite cells to existing myofibers. We reported previously that 0–14 day intermittent feeding led to persistent retardation in myofiber hypertrophy. However, how satellite cells respond to such nutritional insult has not been adequately elucidated.ad libitum feeding), intermittent feeding  and re-feeding  groups. Chickens were killed on Day 15 and satellite cells were isolated. When cultured, satellite cells from the IF group demonstrated significant retardation in proliferation and differentiation potential, while RF partly restored the proliferation rate and differentiation potential of the satellite cells. Significant up-regulation of insulin like growth factor I receptor (IGF-IR) (P<0.05) and thyroid hormone receptor α (TRα) (P<0.05), and down-regulation of growth hormone receptor (GHR) (P<0.01) and IGF-I (P<0.01) mRNA expression was observed in freshly isolated IF satellite cells when compared with Con cells. In RF cells, the mRNA expression of IGF-I was higher (P<0.05) and of TRα was lower (P<0.01) than in IF cells, suggesting that RF restored the mRNA expression of TRα and IGF-I, but not of GHR and IGF-IR. The Bax/Bcl-2 ratio tended to increase in the IF group, which was reversed in the RF group (P<0.05), indicating that RF reduced the pro-apoptotic influence of IF. Moreover, no significant effect of T3 was detected on cell survival in IF cells compared with Con (P<0.001) or RF (P<0.05) cells.One-day-old broiler chicks were allocated to control (Con, 3, and that RF partially reverses these effects. Thus, a moderate nutritional strategy for feed restriction should be chosen in early chick rearing systems.These data suggest that early-age feed restriction inhibits the proliferation and differentiation of satellite cells, induces changes in mRNA expression of the GH/IGF-I and thyroid hormone receptors in satellite cells, as well as blunted sensitivity of satellite cells to T However, we found this kind of “feed restriction” could have a long-term negative influence in skeletal muscle growth if endured during the early growth period (0–14d)thymidine[Previous studies regarding the nutritional influences on satellite cells have been focused on the changes of cell mitotic activity by determining DNA synthesis with incorporation of either hymidine,10 or Brhymidine,11,12,27We found that 2 days re-feeding after 12 days of intermittent feeding was unable to restore completely the proliferation and differentiation capabilities of the satellite cells, as indicated by the cell morphology and viability detected with the MTT assay. This result adds to the previous findings that several days of re-feeding was able to completely reverse the depressed mitotic activity of the satellite cell caused by short-term (2–3 days) feed deprivation or fasting in chicks or turkeys,12,27,28Previous findings have shown a role for apoptosis in muscle induced by under-nutrition-30, so hIt is suggested that the GH/IGF-I system mediates the effect of nutritional state on satellite cells. Feed rein vitro[in vivo[in vitro[The role of the GH/IGF-I axis in the regulation of avian muscle growth remains obscure. Growth in vitro,14 and io[in vivo,41, and o[in vivo,14. Sateo[in vivo. IGF-I s[in vitro-46. Howe[in vitro. Since b[in vitro.4 in rats would stimulate the number of total satellite cells and satellite cells per muscle fiber[3 and T4 decreased with IF for 14 days in chicks[3, may be blunted. This speculation was supported in the T3 challenge test for satellite cells from the three different groups  immediately after hatching impairs proliferation and differentiation capabilities of satellite cells, which could not be completely restored by 2 days of re-feeding. The disrupted satellite cell viability was associated with alterations in mRNA expression of the GH, IGF-I and thyroid hormone receptors, as well as the blunted sensitivity of satellite cells to Teviously can be eThe authors declare that they have no competing interests.YL carried out the experiments, participated in the data collection, data analysis and interpretation, and drafted the manuscript. XY and YN helped in data analysis and interpretation, and paper drafting. ED, JB, NE and RG provided valuable advice for this study and helped in editing the manuscript. RZ contributed in conception, experimental design, data interpretation and finalized the manuscript. All authors read and approved the final manuscript.


Parasite infections often result in a switch of the human body’s predominant immune reaction from T-helper 1 (Th1)-type to Th2-type. Hence, parasite infections are widely expected to accelerate the progression of human immunodeficiency virus (HIV) infections to acquired immunodeficiency syndrome (AIDS). In the People’s Republic of China, both parasitic diseases and AIDS are epidemic in certain rural areas, and co-infections are relatively common. However, no population-based studies have yet investigated the frequency of HIV and parasite co-infections, and its effects on immune responses. We studied (1) the immune status of an HIV-infected population, and (2) the effect of co-infection of HIV and intestinal parasites on selected parameters of the human immune system.A total of 309 HIV-infected individuals were recruited and compared to an age-matched and sex-matched control group of 315 local HIV-negative individuals. Questionnaires were administered to all participants to obtain information on sociodemographic characteristics, sanitation habits, family income, and recent clinical manifestations. Two consecutive stool samples and 10 ml samples of venous blood were also collected from each individual for the diagnosis of parasite infections and quantitative measurements of selected cytokines and CD4+ T-lymphocytes, respectively.Blastocystis hominis (B. hominis) (13.9%) and Cryptosporidium spp. (10.1%). The prevalence of Cryptosporidium spp. in HIV-infected individuals was significantly higher than that in HIV negative individuals (P < 0.05). Compared to the non-co-infected population, no significant difference was found for any of the measured immunological indicators (P > 0.05). However, the following trends were observed: IFN-γ levels were lower, but the IL-4 level was higher, in the population co-infected with HIV and helminths. In the population co-infected with HIV and B. hominis, the IL-2 level was higher. The population co-infected with HIV and Cryptosporidium spp. had markedly lower CD4+ T-lymphocyte counts.During the study period, 79 HIV-infected individuals were not under highly active antiretroviral therapy (HAART) and were thus included in our analysis; the prevalence of intestinal helminth infections was 6.3% and that of protozoa was 22.8%. The most common protozoan infections were Cryptosporidium spp. was not associated with a significant change in immune factors but co-infection with Cryptosporidium spp. was associated with a reduced level of CD4 + T-lymphocytes, confirming the opportunistic nature of such infections. Co-infection with B. hominis, on the other hand, was associated with an antagonistic shift in the immunological profile compared to an HIV infection.According to the immunologic profile, co-infection with helminths is disadvantageous to HIV-infected individuals. It was associated with a shift in the Th1/Th2 balance in the same direction as that caused by the virus itself, which might indicate an acceleration of the progress from an HIV infection to AIDS. Co-infection with  Please see Additional file Cryptosporidium spp., microsporidia, Giardia intestinalis, and Strongyloides stercoralis) [HIV/AIDS is a global health problem , as are coralis) -12. HIV coralis) ,14. On tcoralis) . In addicoralis) -18.Immunological studies have shown that T-helper 1 (Th1)-type immune responses are associated with viral infections  while thIn the People’s Republic of China (P.R. China), both intestinal parasitic diseases and AIDS are endemic in rural areas -26. TherThe study was conducted in Fuyang in the Anhui Province in P.R. China between June and August 2008. The criteria for participants were: (1) 6–65 years of age, (2) ability to give written informed consent or to obtain assent by legal guardians, and (3) absence of obvious mental illnesses or other diseases affecting investigation procedures.HIV-infected individuals registered with local and provincial health departments were contacted for inclusion in the study. A control group of HIV-negative individuals residing in the same communities was then identified, matched by age and sex. The control group was recruited from family members of HIV-infected study participants and, if no suitable controls were available, from people in their neighborhood.Questionnaires were administered to all study participants by health professionals from the local Center for Disease Control and Prevention (CDC). All interviewers had training for routine tasks related to HIV care and prevention. Questions focused on sociodemographic characteristics, sanitation habits, family income, and recent clinical manifestations. One fresh stool samples was collected from each study participant over several days, with a second sample collected whenever possible to maximize the sensitivity of the diagnostic approaches (maximum one sample/day). A 10-ml venous blood sample was also obtained from each study participant.Current HIV – and intestinal parasite infection status, cytokine concentration, and CD4+ T-lymphocyte counts – were determined for all study participants. HIV infections were diagnosed using an enzyme-linked immunosorbent assay . The critical cutoff value was calculated as the mean optical density (OD) value of the positive controls multiply by 10%. Individuals with an OD value below the critical value were considered HIV negative. ELISA-positive samples were subject to confirmation by Western blot . Individuals with two positive test results were confirmed as HIV positive. Both tests were conducted in the local CDC station.The CD4+ T-lymphocyte counts were determined using FACSCalibur flow cytometry . Quantitative ELISA kits  were used to measure cytokine concentrations in the serum. Tested cytokines included IL-2, IL-4, IL-10, and IFN-γ. Both CD4+ T-lymphocyte and cytokine measurements were completed by staff of the National Institute of Parasitic Diseases in Shanghai and the Institute of Parasitic Diseases of the Anhui Province.Ascaris lumbricoides, hookworm, Trichuris trichiura), Clonorchis sinensis, and Strongyloides stercoralis. Eggs of the former four species were identified morphologically by examining three Kato-Katz slides from each stool sample while S. stercoralis larvae were detected by the charcoal culture method. For the diagnosis of protozoa, an in vitro culture method [B. hominis while Cryptosporidium spp. was detected by modified acid-fast staining [G. intestinalis and Entamoeba spp. were detected by the Lugol’s iodine method [The Kato-Katz  and chare method  was emplstaining . G. intee method . A positt-test, Dunnett t test, variance analysis, and χ2 testing were used for statistical data analysis in SAS9.1, as appropriate.All data were entered twice into a database established using EpiData 3.1. The Student’s An optimized immune function model (1)  was usedT is defined as the proportion of cytokines associated with Th1-type cells in the total cytokine pool. IFN-γ is considered as an indicator of Th1 responses and IL-4 as an indicator of Th2 responses.Here, The study protocol was approved by the institutional review board of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention in Shanghai. Potential study participants were contacted through the village leaders, and the objectives, procedures, and potential risks were carefully explained. Interested individuals provided written informed consent in person or, in the case of minors, assent was granted by parents or legal guardians before enrolment into the study. All participants were offered professional counseling before and after HIV testing by staff of the local AIDS prevention and treatment agencies where they were enrolled for free treatment and follow-up. Free anthelmintics  were offered to all participants found to be infected with helminths through local healthcare institutions. All diagnostic results and other data collected in the frame of the study were strictly confidential.A total of 624 individuals were enrolled into the study. Among them, 590 individuals  provided samples of sufficient quality and quantity to perform all three examinations, i.e. parasite diagnosis in stool, and cytokine and CD4+ T-lymphocyte quantification in blood  and 10.1% (8/79), respectively. Three individuals were infected with both B. hominis and Cryptosporidium spp.Among the 79 HIV-infected individuals who were not on HAART, the overall prevalence of helminth infections was 6.3% (5/79) and that of protozoa infection was 22.8% (18/79). B. hominis in the HIV-negative population was markedly higher (21.8%) than that in the HIV-infected population (13.9%), whereas that of Cryptosporidium spp. was significantly lower in the HIV-negative population (3.1%) compared to the HIV-infected population (10.1%) (P = 0.0134). The parasite infections in the HIV-positive and -negative populations are shown in Table Among the 294 HIV-negative individuals, 5.4% 16/294) were infected with helminthes and 24.8% (73/294) with protozoa. Both prevalences were not significantly different from the recorded value among the HIV-infected population. Five individuals were infected with both helminths and protozoa. The prevalence of 6/294 werT value of the HIV-positive group (0.35) is significantly lower than that of the in HIV-negative group (0.45). The pattern remained the same if participants with parasitic infections were excluded from the analysis  compared to their HIV-negative counterparts. Among the individuals co-infected with HIV and helminths or Pneumocystis spp., microsporidia, and Cryptosporidium spp. [Cryptosporidium spp. in the HIV-infected population was 10.1%, significantly higher than that of 3.1% among HIV-negative group. The immune profile of HIV-infected individuals was markedly different from that of their HIV-negative peers. However, no significant change in immune system indicators was observed when individuals with HIV infections but no parasite co-infection were compared to those with such co-infections. Cryptosporidium spp. infections in HIV-infected individuals often cause severe diarrhea and malnutrition, and can even lead to death among AIDS patients [It is now widely accepted that by undermining the human immune response, HIV infections facilitate the establishment of opportunistic parasite infections such as ium spp. -35. Indepatients .B. hominis is one of the most common human intestinal parasite infections. B. hominis is usually considered a commensal (non-pathogenic) protozoa inhabiting the human intestine, although previous studies have suggested that it has certain pathogenicity [B. hominis among HIV-infected individuals was actually lower than that among the controls in our study.genicity -39. InteB. hominis co-infection, CD4 levels were slightly lower compared to individuals without a B. hominis infection, while IFN-γ and other cytokine levels did not change significantly and IL-2 levels were higher. IL-2 is indispensable for cell-mediated immune responses against parasitic infections and humoral immunity. Therefore, the presence of B. hominis may even be favorable for HIV-positive individuals as IL-2 induces T cells to secrete IFN-γ, and the latter is an important factor in anti-viral immune reactions. The promotion of human B cell proliferation should inhibit IL-4. However, in our study IL-4 levels were found to be slightly higher. To sufficiently determine the reasons, further studies need to be conducted. The IL-10 level was slightly lower if co-infection of HIV and B. hominis was present. IL-10 can inhibit Th1-stimulating cytokine secretion. Together, these findings indicate, once again, a need to further study the pathogenicity of B. hominis in order to better determine its status as a member of the intestinal flora [The appropriate immune reaction against helminth infections is believed to be provided by Th2 cells which inhibit the development of Th1 cells, and hence prevent them and macrophages from mounting massive immune reactions against the comparatively very large parasites – a reaction which would potentially be harmful for the human body ,41. The al flora -47.Cryptosporidium spp. was not associated with any significant change in studied immune factors while co-infection with B. hominis was associated with favorable shifts in the immune profile.In conclusion, co-infection with helminths was associated with potentially detrimental changes of the immunologic profile of HIV-infected individuals. Helminth infections appeared to act synergistically with HIV in shifting the Th1/Th2 balance, a shift which has traditionally be seen as indicative of an acceleration of the progress from HIV infection to AIDS. On the other hand, co-infection with protozoa was imperceptible in the immune response profile: co-infection with The authors declare that they have no competing interests.LGT conceived the study, collected the data and analyzed it, and drafted the manuscript. SL and TPW conceived the project, assisted in the interpretation of the results, and revised the manuscript. MKD and PS revised the manuscript and provided intellectual input for the interpretation of the findings. FFW, JG, XMY, and YCC conceived the project and provided technical support for data collection and analysis. JXC conceived the study and revised the manuscript. All authors read and approved the final manuscript.Multilingual abstracts in the six official working languages of the United Nations.Click here for file


Patients with cancer have a 7- to 10-fold increased risk of developing venous thromboembolism. Circulating microvesicles could be a useful predictive biomarker for venous thromboembolism in cancer. Validated and standardised techniques that could be used to determine the complete microvesicle phenotype are required.These were two-fold: a) to characterise tissue factor (TF)-bearing microvesicles released by cultured breast cancer cells MDA-MB-231 by flow cytometry (FCM), transmission electron microscopy (TEM) and thrombin generation assay (TGA); and b) to validate the sensitivity and variability intra/inter-assay of TGA as a useful method to study the procoagulant activity (PCA) of microvesicles.Cultured breast cancer cells MDA-MB-231 were incubated for 45 minutes at 37°C. Samples were then centrifuged or not at 4,500 g for 15 minutes, and cells and MVs or MV-containing supernatants were used for TEM, FCM and TGA. In activity assays, microvesicles (i.e. cell-depleted supernatants) were incubated with anti-TF antibodies or with annexin V to assess the contribution of TF and phospholipids to the PCA. Alternatively, supernatants were filtered through 0.1, 0.22, 0.45 or 0.65 µm membranes and subjected to TGA.The majority of the PCA was associated with microvesicles smaller than 0.1 µm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 121±54 nm with a majority of vesicles between 100 and 200 nm. Microvesicles derived from 5,000 MDA-MB-231cells/ml were sufficient to significantly increase the thrombin generation of normal pooled plasma.TEM, FCM and filtration coupled to TGA represent a useful combination to study the PCA of TF-bearing microvesicles, whatever their size. And it will be interesting to implement these techniques in patients. Venous thromboembolism (VTE) is responsible for 15% of deaths in cancer patients . TherefoIt is therefore essential to validate and standardise a panel of techniques that could be used to determine the complete MVs phenotype and to study the effects of pre-analytical steps on MVs’ conformation .The objectives of this study were therefore to (a) to characterise tumour-cell-derived MVs released by cultured MDA-MB-231 breast cancer cells with FCM, transmission electron microscopy (TEM) and TGA; and (b) to validate the intra/inter-assay sensitivity and variability of TGA as a useful method to analyse MVs PCA.2 humidified atmosphere at 37°C. Natrium bicarbonate was set at 1.5 g/L to control the pH level. Cell viability was controlled by the trypan blue exclusion test.Adherent MDA-MB-231 breast cancer cells (ATCC number HTB-26) were obtained from the American Type Culture Collection  and were cultured in RPMI-1640 medium  supplemented with 10% foetal bovine serum (FBS) . Cells were maintained in a 5% CONormal pooled plasma (NPP) was prepared as previously described . PlateleA modified version of the protocol reported by Davila et al. was performed . Cells w®), mounted on a grid and stained with heavy metal salts (uranyl acetate and lead citrate). The samples were observed using Tecnai 10 TEM . The MV size determination was performed semi-automatically on high magnification TEM pictures  on MDA-MB-231 cells and MVs were carried out by FCM as mentioned in supplementary information. MUC-1 was chosen since it has been shown that TF-MV activity of patients with breast cancer who presented with acute VTE, correlated with the presence in the blood of MV expressing the epithelial antigen MUC1 .PCA was measured by TGA according to a modified version of the method developed by Hemker . Total cAll the experiments were performed in triplicate. The results were expressed as means±SD. All parameters were calculated using software thrombinoscope. The percentage of PCA was calculated on the lagtime reduction by MVs.Variability of TGA was measured according to the previously described method using 80 µL of NPP+20 µL PBS. The assays were conducted in triplicate on 6 independent runs with the same operator. Mean intra-assay variation and inter-assay variability for lagtime and peak were determined. Mean intra-assay variation corresponds to the mean coefficient of variation for the triplicates (n=6). Inter-assay variation corresponds to the coefficient of variation for the mean value of triplicates between the 6 independent runs.MV fractions were filtered through 0.1, 0.22, 0.45 or 0.65 µm membranes  following the manufacturer's instructions. Supernatant (Sup) and filtrates (Filt) were compared by TGA and FCM (1 experiment in triplicate).For contribution of TF, MVs derived from 600,000 cells/ml were pre-incubated with various monoclonal antibodies (mAbs)  for 20 minutes at room temperature before TGA was performed.The clone HTF-1  was used as TF activity neutralizing Ab . The cloThe phospholipid-mediated activity was blocked with annexin V  . SamplesMVs derived from 100,000 cells/ml were separated from the cells by centrifugation at 4,500 g during 15 minutes. The collected supernatants, which contain only MVs, were ultracentrifuged at 100,000 g during 90 minutes to pellet the MVs. MVs before ultracentrifugation (Sup), pellet of MVs (Pellet UC) and supernatant after ultracentrifugation (Sup UC) were tested in TGA.The standard protocol described above for the in vitro generation of MVs is described in Three centrifugation protocols to eliminate cells were also compared using TGA at 600,000 cells/ml: (a) simple centrifugation at 4,500 g for 15 minutes ; (b) douTo determine the sensitivity of TGA, the impact of cell concentration on TGA was assessed at 100; 500; 1,000; 2,500; 5,000; 10,000; and 20,000 cells/ml. Three independent experiments were performed in triplicate.Comparison between different conditions was performed using the Kruskal–Wallis test on MedCalc software (version 12.2.1.0). If the Kruskal–Wallis test is positive (p<0.05), then MedCalc performs a test for pairwise comparison of subgroups according to Conover .Fig. S1). Ten percent of all MVs were positive for TF expression, and only 1.7% were positive for MUC1  significantly boosted up the generation of active thrombin when compared to the PBS control curve a, as shoSince FCM cannot detect vesicles smaller than 300 nm, and since TEM showed a majority of MVs smaller than 300 nm, the link between the MVs size and PCA was evaluated through filtration of the supernatant using membranes with various cut-off sizes ranging from 0.65 to 0.1 µm b. As comThe use of HTF-1, an anti-TF antibody, was associated with a strong inhibition of the PCA associated to MVs as revealed by a 6-fold increase in lagtime c. On theInclusion of annexin V during the assay was associated with a complete abolishment of PCA d.The majority of PCA contained in supernatant was associated with the pellet after 100,000 g ultracentrifugation, but some activity remained in the post-ultracentrifugation supernatant e: this sThe lagtime and peak intra-assay variation coefficients were 12 and 11%, respectively. The lagtime and peak inter-assay variation coefficients were 14 and 50%, respectively.The stirring of MDA-MB-231 cells during MVs generation does not modify the PCA of cells (Cells) and MVs (Sup) derived from 600,000 cells/ml a. HoweveThe centrifugation protocol used to separate cells and MVs does not significantly impact TGA curves b. IndeedAs shown in Fig. S1). MUC-1 was chosen since it has been shown that TF-MV activity of patients with breast cancer who presented with acute VTE, correlated with the presence in the blood of MVs expressing the epithelial antigen MUC1  and annexin V, which links phosphatidylserine, we confirmed that the PCA of MVs is related to the expression of active TF and negatively charged PL . MoreoveBecause the clinical importance of MVs is increasingly being recognized, it is crucial to standardise methods to allow comparison of data across different studies. To validate the use of TGA for measuring the PCA of MVs, we assessed the impact of 2 pre-analytical variables, that is, stirring and centrifugation, and determined the sensitivity of this technique. Contrary to a previous report , the stiIn vivo the correlation between TF-MV and thrombosis may become more or less strong depending on the type of tumour/cancer present in a patient , 7, 10. In addition, atomic force microscopy, a high-sensitive technique able to image biological samples in aqueous fluids, was recently proposed for the detection and quantification of CD41-positive MVs . This teThis is the first study that uses FCM, TEM and TGA to characterise MVs. MDA-MB-231 releases MVs whose size was 121±54 nm by TEM. TGA is a useful technique to study the PCA of tumour-cell-derived MVs whatever their size. It should be combined by high-sensitive sizing and counting techniques such as AFM in order to estimate the concentration of specific MVs (i.e. TF-MVs). TGA should be validated and used to study TF-MV in plasma from healthy subjects and patients.


Tumor spheres enriched in GSCs derived from the mouse GL261 glioma cell line, and the brain microvessel endothelial cell line, b.END3, were used in this study. A Transwell co-culture system, RNAi experiments, quantitative polymerase chain reaction, western blotting and enzyme-linked immunosorbent, cell counting kit-8 (CCK-8) proliferation, Transwell migration and wound-healing assays were used in this study to determine the migration and proliferation ability, as well as the Hedgehog (HH) pathway-related gene expression in the b.END3 cells. Based on the results, it was demonstrated that the migration and proliferation of the endothelial cells were enhanced following co-culture with GSCs. The gene expression of the HH pathway-related genes, Sonic Hedgehog (Shh) and Hedgehog-interacting protein (Hhip) was altered in the endothelial cells when co-cultured with GSCs. Overexpression of glioma-associated oncogene homolog 1 indicated activation of the HH pathway. Following knockdown of smoothened (Smo) in the endothelial cells, the migration and proliferation abilities of the cells were inhibited. GSCs have little effect on enhancing these behaviors in endothelial cells following Smo-knockdown. Further investigation revealed that Shh levels in the supernatant of the co-culture system were elevated, indicating the importance of secreted Shh from the endothelial cells. In conclusion, GSCs enhanced the migration and proliferation of the endothelial cells  Glioblastoma multiforme (GBM) has been one of the most lethal malignant brain tumors in adults for the past 30 years. The best treatment to date is a combination of surgery, radiation therapy and temozolomide administration; however, this does not produce sufficient results. The overall survival time of patients with GBM is between 26 and 52 weeks –3. Previ3 without new vessels  pathway is extremely important in embryonic development, since it directs embryonic growth and cell fate determination. The canonical HH pathway is activated by HH ligands, mostly by Sonic Hedgehog (Shh) in the nervous system, which binds to receptor complexes, such as the transmembrane protein, Patched, and the G-protein-coupled protein, Smoothened (Smo). Hedgehog-interacting protein (Hhip) is one of the inhibitory ligands that binds to Shh. The binding of Shh to Patched releases Smo. Smo then alters the activity of glioma-associated oncogene homolog 1 (Gli1) protein and initiates the transcription of downstream genes. The HH pathway is expressed in endothelial cells. It has been demonstrated that the proliferation and migration of endothelial cells are associated with the HH pathway .The aim of the present study was to investigate whether GSCs enhance proliferation and migration of endothelial cells, and whether the HH pathway plays a role in this process.The mouse GL261 glioma and brain microvascular endothelial cell lines  were used in this study. The two cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. GSC spheres were obtained from GL261 cells and cultured in serum-free DMEM/F12 medium with 10 ng/ml epidermal growth factor , 10 ng/ml basic fibroblast growth factor  and 2 mg/ml B27  ,12.5 b.END3 cells were seeded in 6-well plates, while 1.5×105 GSCs were seeded in the cell culture chamber. The insert was transfered into the well with endothelial cells after 24 h of culture.For the co-culture system, Transwell cell culture inserts were used  to explore the indirect effect of GSCs on endothelial cells. Briefly, 2×105 cells were seeded in each cell culture insert. Serum-free cell culture medium (400 μl) was added to each insert. One milliliter medium with 5% FBS was added to the 6-well plate, then the insert was transferred to the well and cultured at 37°C. At the end of the migration assay, the insert was washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. The cells that were unable to migrate were wiped out and the rest were stained by crystal violet solution . The cells were counted under a microscope at each field.Transwell cell culture inserts were selected . The cells were trypsinized and counted, then 1.5×104) were seeded in a 6-well plate cultured with DMEM in serum. Pipette tips (200 μl) were used to scratch three parallel vertical lines in each well subsequent to 12 h of culture. The wells were washed with PBS, then the medium was changed to serum-free DMEM. Scratch lines were observed under a microscope and scratch distances were measured, with images captured at 6, 8, 12 and 24 h after scratching.The b.END3 cells  was acquired from the NCBI database. Murine shRNA targeted to Smo was designed, synthesized and packaged with lentivirus by SBO Biomedical Technology . The endothelial cells were seeded in a 6-well plate at 1×103 cells/well, using 40 wells each. Co-cultured medium or control medium (both 150 μl) or 50 μl fresh medium was added to each well subsequent to 12 h of culture. The specific grouping was as follows: 40 wells with NC b.END3 plus medium a, 40 wells with NC b.END3 plus medium b, 40 wells with siR-Smo b.END3 plus medium c and 40 wells with siR-Smo b.END3 plus medium d. At each time-point, the medium in each well was changed to 200 μl fresh medium mixed with 10 μl CCK-8 solution for 10 wells from each group. The absorbance at 450 nm was detected following 2 h of incubation.A CCK-8 proliferation assay kit (Beyotime Institute of Biotechnology) was used in this experiment. A 48-h co-culture medium and a 48-h control well culture medium were collected from the empty control vector  and siR-Smo b.END3 cells (medium c and d). NC b.END3 and siR-Smo b.END3 cells were seeded separately in a 96-well plate at 3×10The total RNA of the b.END3 cells was extracted with RNAiso reagent . Reverse transcription and PCR were performed using a Takara RNA PCR (AMV) kit with primers designed for murine genes. The primers were synthetized by Invitrogen . The sequences of each primer pair and the product size are presented in The proteins of the b.END3 cells were extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology) with the protease inhibitor phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). The proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% milk, then incubated with primary rat anti-mouse Gli1 , rat anti-mouse Shh , rabbit anti-mouse Hhip  and rabbit anti-mouse GAPDH  antibodies overnight at 4°C. The membranes were then washed and incubated with goat anti-rat and goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies  for 2 h at room temperature. The proteins were detected by enhanced chemiluminescence detection reagent. GADPH was used as the loading control.Cell culture supernatant was acquired from the wells of the GSCs, b.END3 cells and the co-culture. Medium was removed and centrifuged at 3,000 × g after 12, 24 and 48 h of culture, then stored at −80°C immediately. An ELISA kit for mouse Shh N-Terminus was purchased from R&D Systems and an ELISA kit for mouse Hhip was purchased from USCN Life Science Inc. . ELISA was performed strictly according to the manufacturer's instructions.All experiments were conducted at least three times and the results were from representative experiments. Data are expressed as the mean ± standard deviation and the statistical significance between the experimental and control groups was analyzed with SPSS 16.0 statistical software . When two groups were compared, the unpaired Student's t-test was used. P<0.05 was considered to indicate a statistically significant difference.in vitro model in order to rebuild an approximate in vivo niche, which we consider to be better than just using a GSC-conditioned medium. In this model, the two types of cells interact via soluble factors, but do not have direct connections. The b.END3 cells were seeded in the lower chambers and GSCs were seeded in the upper chambers. When the wound-healing assay was processed in the co-culture wells, it was clear that the co-cultured b.END3 cells exhibited enhanced migration, since the scratches in the co-cultured wells were narrower than in the control . It has been discovered that the location of GSCs is close to the microvessels . Tumor mThe mechanism of GSC-regulated tumor angiogenesis is a hot topic of research –18. VascThe present study indicates a new mechanism of tumor angiogenesis. The results demonstrated that GSCs regulate the gene expression of nearby endothelial cells, specifically through the HH pathway, thereby affecting their biological behavior. The HH pathway is a classical pathway of cell survival, proliferation and migration, and Shh is one of its classical activators, which functions in autocrine and paracrine ways . The HH Progression has been made in the study and treatment of cancer using anti-angiogenesis drugs. However, adverse effects have been observed. Microvasculature fracture causes local anoxia, which is another niche for CSCs, causing local appearance of the cells. In future studies, we aim to interfere with the interaction between GSCs and microvessels, for example the HH pathway, to inhibit tumor invasion and propagation and to provide opportunities for tumor resection.